Method and apparatus for timing intermittent illumination of a sample tube positioned on a centrifuge platen and for calibrating a sample tube-imaging system

ABSTRACT

Centrifuged material layer volumes are measured and quantified during centrifugation of the material layers contained in a sample tube in a centrifuge assembly, which sample tube is disposed on a centrifuge platen. The material layers in the sample tube are periodically illuminated during centrifugation by a pulsed light source which differentially excites one or more fluorescent dyes or stains which are admixed with the sample being analyzed. This kinetic procedure is particularly useful in performing differential blood cell and platelet counts. A fluorscent target reference device is positioned on the centrifuge platen along with a detectable platen position sensor. The centrifuge assembly includes a processor controller which receives information from the platen position sensor, and from the target reference device, and which controls operation of the light source.

TECHNICAL FIELD

[0001] This invention relates to a method for rapidly making centrifugedmaterial layer volume measurements. The method of this invention isparticularly useful in performing blood constituent count measurementsin a centrifuged sample of anticoagulated whole blood.

BACKGROUND ART

[0002] The measurement of the blood cell counts in a centrifuged sampleof anticoagulated whole blood has been described in the scientificliterature, and a method for easily measuring certain blood cell andother constituent layers is described in U.S. Pat. No. 4,027,660,granted Jun. 7, 1977 to Stephen C. Wardlaw et al. In the patentedmethod, a sample of anticoagulated whole blood is centrifuged in aprecision capillary tube which contains a plastic float. The floatlinearly expands some of the cell layers and the platelet layer.

[0003] In performing the patented method, the blood sample iscentrifuged for about five minutes at about 12,000 RPM, and then theexpanded lengths of the cell and platelet layers are measured. One ofthe problems in the patented method pertains to the relatively highcentrifuge RPM required to reliably compact the layers, particularly theplatelet layer. If the constituent layers are not completely oruniformly packed, the results derived by the method may be inaccurate.The aforesaid high RPM centrifugation requires an expensive centrifuge,and increases the risk of tube breakage. Another problem relates to therequired minimum five minute centrifugation time period, which isundesirable in many medical situations. Still another problem relates tothe need for the operator to remove the tube from the centrifuge andre-insert it into a reader. Because this operation must be performedwithin a limited time interval following centrifugation, it requires theclose attendance of the operator, which is inefficient and furtherexposes the operator to a potentially hazardous sample.

[0004] It would be desirable to be able to measure the blood constituentlayers in a shorter period of time, and with a lower RPM centrifuge,and/or to reduce the amount of sample tube handling.

DISCLOSURE OF THE INVENTION

[0005] This invention relates to an assembly for quickly determiningindividual material volume measurements during centrifugation of agravimetrically separable material mixture sample, such as ananticoagulated whole blood sample. Additionally, this invention relatesto a method which can determine blood constituent volume measurements,and blood constituent counts during centrifugation of an anticoagulatedwhole blood sample before the centrifugation step is finished. Themethod of this invention utilizes a combined centrifuge and reader whichwill perform the functions of both centrifugation and reading, and thussimplify the performance of the material layer measurements described inthe aforesaid U.S. patents. The method of this invention provides akinetic analysis of blood cell compaction. By following the principlesof this invention, white blood cell and platelet layers can bequantified in a relatively low RPM centrifuge, for example a centrifugewhich operates at speeds of between about 8,000 to about 10,000 RPM,although the higher speed 12,000 RPM centrifuge can also be used.

[0006] During the process of gravimetrically forming cell layers in acentrifuged anticoagulated whole blood sample there are twocounteractive forces at work, i.e., outward compaction of the cells andother formed components in the sample; and inward percolation of theplasma in the sample. As the cells and other formed component layers inthe blood sample settle during centrifugation, the layers compactthereby decreasing the length of the layers. At the same time, the fluid(plasma) component of the blood sample percolates through the compactinglayers.

[0007] For cell compaction to occur, the fluid component must bedisplaced, but as the cell layers continue to compact the percolationpaths for the fluid become more tortuous. Cell layer compaction willinitially progress rapidly but will become increasingly slower ascompaction increases and percolation becomes more difficult. Thus therate of compaction is non-linear. Since the rate of compaction variesconsiderably between different blood samples due to fluid viscosityand/or other factors, a single reading of a cell layer which is takenprior to complete compaction of the cell layer cannot be extrapolated topredict the extent of the final cell layer compaction. Therefore thethickness (or length) of the fully compacted cell layer cannot bedetermined from a single measurement of the thickness (or length) of acell layer which reading is taken during the centrifugation step. Thisfact has formed the basis of the traditional method of determining theoptimum centrifuge speed, and time, so that measurements of the celllayers' thickness will be made only after the cell layers would beexpected to show no further compaction. “This complete compaction”centrifugation time is considered deemed to be the minimum time requiredbefore measuring any centrifuged anticoagulated whole blood constituentlayers.

[0008] I have discovered that the inherent unpredictability of theextent of final blood cell layer compaction can be overcome by makingseveral separate preliminary measurements of the cell layers' thicknessduring the ongoing centrifugation step, and then fitting the deriveddata to a non-linear mathematical algorithm that will predict the fullycompacted cell layer thickness. This procedure does not require ultimatelayer compaction, and in fact, the fully compacted cell layer thicknessmay be mathematically predicted after only four or five preliminary celllayer thickness measurements. Additionally, the method of this inventionallows for an accurate calculation of the ultimate extent of blood celllayer compaction, and therefore the thickness of a fully compacted celllayer, over a wide range of centrifuge speeds Thus a plurality of celllayer thickness measurements that are taken during the low speedcentrifugation of an anticoagulated whole blood sample can accuratelypredict the ultimate degree of cell layer compaction which will resultfrom a prolonged centrifugation step that is performed at a much highercentrifuge speed, such as is required by the prior art.

[0009] The method of this invention involves the use of: a centrifuge; afluorescent colorant excitation light source; a photodetector; and amicroprocessor controller for controlling operation of the assembly, andfor collecting data from readings taken by the assembly. The lightsource is preferentially a high intensity pulsed light source whichperiodically illuminates the blood sample in the sampling tube as thelatter is being centrifuged. Illumination of the blood sample in thetube causes fluorescence of certain of the blood cell constituents aswell as illumination of the red blood cell layer, so that thephotodetector can discriminate between the various cell layers in thetube that are gravimetrically compacted during the centrifugation step.By selection of the appropriate filters on both the light source and thedetector, the light reflected from the material layers can be measured,as well as the fluorescence. The pulsing of the light source issynchronized with the position of the tube during centrifugation, sothat the tube will be illuminated as it passes by the photodetector. Theoptics and filters used in performing the method of this invention aregenerally similar to those described in U.S. Pat. No. 4,558,947, grantedDec. 17, 1985 to S. C. Wardlaw, the disclosure of which patent isincorporated herein in its entirety.

[0010] The light source for Illuminating the tube for the excitation offluorescence must have sufficient emission in the excitation band (about420-480 nm) to provide adequate emission energy from the sample tube,when received by a filtered, solid-state image dissector, such as a CODarray. Further, the energy must be delivered in the period when the tubeto be imaged is within the focal range of the detector, which istypically about 50 μsec. These requirements are best met by a xenonflash tube having an associated focusing means, rather than a diffuser,which flash tube is driven by a power supply capable of delivering shortpulses at the needed power levels and wherein the light flashes areprecisely tuned to the position of the tube relative to the detectorwhen the flash tube is triggered.

[0011] If the sample is analyzed by the aforesaid kinetic technique, theextent of compaction of the several blood sample constituent layers isperiodically imaged during centrifugation, and the sequentialconstituent layer images are stored in the microprocessor controller inthe assembly. After a sufficient number of images have been obtained andstored, about four or five for example, the microprocessor controllerwill be able to calculate the degree of ultimate compaction of theconstituent layer or layers being measured, and will display thecalculated value. At that point, centrifugation of the sample will beterminated. The microprocessor controller controls operation of theassembly in that, on command, it will: initiate centrifugation; monitorthe RPM of the centrifuge; synchronize the light pulses with the ongoingcentrifuge RPM; control operation of the photodetector; receive andstore constituent layer readings; calculate the ultimate degree ofconstituent layer compaction, and the resultant constituent counts orvalues; and shut the centrifuge down. The operator thus need only placethe blood sampling tube in the centrifuge, and initiate operation of theassembly. For operator convenience and safety, the blood sampling tubecan be contained in a special cassette of the general type described inU.S. Pat. No. 5,776,078.

[0012] If, on the other hand, the object is to measure the final extentof cell compaction following a fixed period of centrifugation, the layerlengths may be analyzed by the taking of one or more images followingthe fixed period centrifugation while the centrifuge continues spinning.Thus the advantage of being able to measure the extent of cell layercompaction without having to transfer the blood sample tube from thecentrifuge to a separate reader instrument Is realized. A device forsynchronizing the pulsing of the light source irregardless of the speedof rotation of the centrifuge is also provided in the assembly. Such adevice takes into account centrifuge wear, eliminates the need to set oradjust the pulsing synchronization of the assembly, and also allows thecentrifuge to be intentionally operated at different speeds.

[0013] It is therefore an object of this invention to provide a methodfor deriving an ultimate material layer thickness measurement in acentrifuged material mixture prior to the actual achievement of theultimate layer thickness.

[0014] It is another object of this invention to provide a method whichwill allow the reading of the material layer thicknesses following afixed period of centrifugation while the sample is still within thecentrifuge.

[0015] It is a further object of this invention to provide a method ofthe character described wherein the material mixture is ananticoagulated sample of whole blood.

[0016] It is an additional object of this invention to provide a methodof the character described wherein a plurality of preliminary sequentiallayer interface locations are sensed and stored during centrifugation ofthe mixture, with the interface data obtained being used to calculatethe ultimate layer thickness.

[0017] It is a further object of this invention to provide a method ofthe character described wherein the ultimate thickness of a plurality ofmaterial layers in the sample being centrifuged can be derived frompreliminary layer thickness measurements.

[0018] These and other objects and advantages of the invention willbecome more readily apparent from the following detailed description ofthe invention when taken in conjunction with the accompanying drawings,in which:

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is a schematic view of a tube containing a material mixturesample as the latter is being centrifuged in the tube;

[0020]FIG. 2 is a plot of the dynamics of layer compaction duringcentrifugation of the material mixture, with the layer thickness beingplotted against elapsed centrifugation times;

[0021]FIG. 3 is a graph showing how an ultimate platelet layer heightwas calculated in a specific case when a blood sample was centrifuged attwo different speeds, with platelet layer being plotted against thereciprocal of elapsed centrifugation times;

[0022]FIG. 4 is a schematic perspective view of a blood samplemeasurement assembly used in performing the method of this invention;

[0023]FIG. 5 is a perspective view of a preferred embodiment of acentrifuge platen and drive mechanism designed for use in connectionwith this invention;

[0024]FIG. 6 is a fragmented perspective view of a tube holder and tuberotating mechanisms which are utilized in performing the method of thisinvention;

[0025]FIG. 7 is a sectional view of the tube rotating mechanism used inperforming the method of this invention; and

[0026]FIG. 8 is a plan view of the centrifuge platen showing the lightpulsing synchronizing and imaging system calibration feature of theassembly.

DETAILED EXAMPLE FOR CARRYING OUT THE INVENTION

[0027] Referring now to FIG. 1, there is shown a schematic view of atube 2 having a transparent side wall 4 and closed at the bottom. Thetube 2 is filled with a mixture of a particulate material componentsuspended in a liquid component 6. FIG. 1 illustrates the dynamics ofparticulate compaction and liquid percolation, as indicated by arrows Aand B respectively, both of which occur during centrifugation of theparticulate component/liquid component mixture. As centrifugationprogresses, the particulate component will gravimetrically separate fromthe liquid component 6, and will, at some point in time, form aninterface 8. The interface 8 continually gravitates away from the uppersurface 7 of the liquid component 6 as centrifugation continues. It willbe understood that more than one interface 8 may form in the samplebeing centrifuged, depending on the nature of the sample.

[0028] I have discovered that when the position of the particulatecomponent/liquid, or component/component interfaces 8 are monitored overthe course of time, the sequential positions of the interfaces define afunction curve 10 of the general type depicted in FIG. 2, wherein theinitial change in interface position is relatively rapid, but slowsappreciably as particulate component compaction progresses. At somepoint in time, particulate component compaction ceases, and theparticulate component-liquid component separation is complete. In thecontext of this invention, I refer to this cessation of particulatecomponent compaction as “ultimate” compaction which is represented bythe dotted line 11 in FIG. 2.

[0029] It will be noted that the same phenomenon occurs when a complexmixture of particulate components suspended in a liquid, such asanticoagulated whole blood, is centrifuged. In this case a plurality ofparticulate component layers will form gravimetrically, with the liquidcomponent 6 percolating to the top of the centrifuge tube 2. Blood is anexample of such a complex mixture of particulate components, since thered blood cells are heavier than the granulocytes, lymphocytes,monocytes and platelets, in order of decreasing density, and since allof the cells in a blood sample are heavier than the plasma component ofthe blood sample. When an anticoagulated sample of whole blood iscentrifuged the various cell/cell and cell/plasma interfaces willgravitate through the blood sample in the same general manner as isillustrated by

[0030]FIG. 2. It should be noted that in a complex mixture of materials,such as whole blood, under some circumstances middle layers may actuallyincrease in thickness rather than compact. This may occur when theseparation of a target component from the mixture exceeds the compactionrate of that layer. In all cases, however, the mathematical analysis andextrapolation is performed identically to, and has the same effect asthat done on the layers which decrease in thickness.

[0031] The curve described by the rate of change of the location of anyparticulate constituent interface, and therefore constituent layerthicknesses in a sample undergoing centrifugation is essentially anhyperbola. This fact can be used to mathematically predict the ultimateextent of particulate constituent layer compaction, and therefore theultimate layer thicknesses and layer volumes of the particulateconstituent layer or layers in the centrifuged sample.

[0032] It should be noted that the compaction of the cell layers may notimmediately follow a hyperbolic function starting at time zero. Todetermine when the movement of the interface has reached the point whereit follows a hyperbolic function and the ultimate compaction maytherefore be calculated, it is only necessary to periodically monitorthe interface position or layer thickness and calculate successive slopemeasurements as S=dP/(1/t), where: S is the slope of the change; dP isthe change in interface position (or layer thickness); and t is theelapsed time since the start of centrifugation. When successive valuesof S no longer change, subsequent preliminary data points may then becollected and the ultimate layer compaction can be determined.

[0033]FIG. 3 shows a specific example of the same anticoagulated wholeblood sample centrifuged at two different speeds, wherein the slopes ofthe hyperbolic curves as shown in FIG. 2, are linearized by plotting thereciprocal of successive elapsed centrifugation times against successivemeasured thicknesses of the compacted platelet layer interfaces 8 at twodifferent centrifuge speeds, i.e., at 8,000 RPM and 12,000 RPM. The line14 generally denotes the rate of change in the platelet layer thicknessduring centrifugation of the test samples in a high speed of 12,000 RPMcentrifuge; and the line 12 generally denotes the rate of change in theplatelet layer thickness during centrifugation of the test samples in alower speed of 8,000 RPM centrifuge, the latter of which provides only40% of the G-force of the former.

[0034] In order to determine the rate of change slopes, a series ofpreliminary successive platelet layer thicknesses 16 are measured, andleast-squares fits are calculated thereby giving the best path 18amongst the preliminary layer thickness data points 16. To calculate theultimate compacted layer thickness, the regression function isextrapolated to an ultimate spin time, in this case infinity, where thevalue of the reciprocal of the elapsed centrifuge time is zero. Theintercept of the rate of change of layer thickness plot at that point onthe Y axis identifies the ultimate compacted layer thickness. It isnoted that although the centrifuge speeds are considerably different,the end result is the same; and that readings need be taken for only arelatively short period of time, such as for two to three minutes ascompared to five to ten minutes, required by the prior art. Thepreliminary measurements may be taken while the centrifuge continues tospin.

[0035] Referring now to FIG. 4, there is shown a schematic view of acombined centrifuge and reader assembly, which is indicated generally bythe numeral 1. The assembly 1 includes a centrifuge platen 3 whichincludes a recess 5 for holding a transparent capillary tube 9. The tube9 may be placed directly in the recess 5, or the capillary tube 9 may beheld in a cassette (not shown) of the type disclosed in co-pendingpatent application U.S. Ser. No. 08/755,363, filed Nov. 25, 1996. In anyevent, at least one surface of the tube 9 must be optically visualizedfor the purpose of collecting the desired optical information from thetube contents. The centrifuge platen 3 is rotationally driven by a motor13, which is controlled by output line 21 from an assemblymicroprocessor controller 17. The rotational speed of the motor 13 ismonitored by the controller 17 via line 19 thereby allowing thecontroller 17 to regulate the speed of the motor 13 and thus thecentrifuge platen 3. When the centrifuge platen 3 reaches itspredetermined operating speed, which may be between about 8,000 and12,000 RPM, the action of the controller 17 will depend upon the type ofanalysis required. If it is desired to read the material layercompaction following a fixed period of centrifugation, the controller 17will energize the motor 13 for a desired fixed period of time and thelayer compaction reading will be taken thereafter while the centrifugecontinues to spin.

[0036] On the other hand, if it is desired to kinetically measure thematerial layer compaction, multiple sequential readings of thegravitationally compacting blood cell layer heights in the tube 9 aretaken. As the platen 3 rotates, an indexing device 15 on the side of theplaten 3 passes by and interacts with a sensor 23 which sends a signalthrough line 20 to a programmable delay 22. The indexing device 15 canbe a permanent magnet and the sensor 23 can be a Hall-effect sensor.Alternatively, the indexing device 15 could be a reflective member onthe edge of the platen 3, and the sensor 23 could be an infraredemitter-receiver pair. Still another alternative sensing device couldinclude a sensor in the driving motor 13 provided that the platen 3 wererigidly affixed to the shaft of the driving motor 13.

[0037] After a predetermined time has elapsed from the receipt by thecontroller 17 of a signal from the proximity sensor 23, a flash driver24 triggers a flash tube 26 that delivers a brief pulse of light,preferably less than about fifty microseconds in duration. A filter andlens assembly 28 focuses the light of the desired wavelength from theflash tube 26 onto the tube 9. When the flash tube 26 is positionedbeneath the platen 3, the latter will include an opening 3′ between thesample tube 9 and the flash tube 26 and filter and lens assembly 26. Dueto the fact that the exact rotational rotational speed of the platen 3is monitored by the controller 17 via line 19, and since thecircumferential distance between the position of the index 15 and theposition of the tube 9 is fixed, the required time delay for timelyenergizing the flash tube 26 can be determined by the controller 17 andexpressed through data bus 30 so as to control operation of the flashdriver 24.

[0038] When the tube 9 is illuminated by the flash tube 26, the lightreflected by the cell layers, or light from fluorescence of the celllayers, is focused by a lens assembly 32 through a light filter set 34onto a linear image dissector 36, which is preferably a charge-coupleddevice (CCD) having at least 256 elements and preferably 5,000 elementsso as to achieve optimum optical resolution. Light of an appropriatewave length can be selected by an actuator 38 such as a solenoid orstepping motor and which is controlled by the controller 17 via line 40whereby the actuator 38 can provide the appropriate filter from thefilter set 34, depending upon the light wavelength selected by thecontroller 17. Alternatively, electrically variable filters could beused to provide the proper light wavelengths, or CCDs with multiplesensors, each with its own particular filter could be used. Suitableelectrically variable filters can be obtained from Cambridge Researchand Instrumentation, Inc. of Cambridge, Mass. Suitable CCD's areavailable from Sony, Hitachi and others, and are common instrumentationcomponents.

[0039] Just prior to receiving the light flash from the flash tube 26,the electronic shutter in the CCD 36 is opened by the controller 17, vialine 42. Immediately following the flash, the data from the CCD 36 isread into a digitizer 44 which converts the analog signals from each ofthe CCD cells into a digital signal. The digitized data is thentransferred to the controller 17 through a data bus 46 so that the datacan be immediately analyzed or stored for future examination in thecontroller 17. Thus at any desired point during the centrifugationprocess, optical information from the sample tube 9 can be collected andanalyzed by use of the assembly described in co-pending U.S. patentapplication Ser. No. 08/814,536. The mathematical calculations describedabove which can be used to derive “ultimate compaction” can be made bythe controller 17.

[0040] Referring now to FIG. 5, there is shown a preferred embodiment ofa centrifuge platen assembly 3 which is designed for use when theflashing light source 26 and the digitizer 44, which are shown in FIG.4, are positioned above or below the platen 3. The platen 3 is generallydish-shaped and includes an outer rim 50 and a basal floor 52. A centralhub 54 is secured to the platen floor 52, and the hub 54 is closed by acover 56. The hub 54 has a pair of diametrically opposed windows 58formed therein. The sample tube 9 is mounted on the platen 3. One end ofthe tube 9 is inserted into an opening in the hub 54, and the other endof the tube 9 is lowered into a slot 60 formed in a block 62 that ismounted on the platen rim 50. The platen floor 52 is provided with anopening (not shown) covered by transparent plate 64. A counterweight 66is diametrically disposed opposite the plate 64 so as to dynamicallybalance the platen 3. The plate 64 allows the platen 3 to be used withlight sources and detectors which are located either above or below theplaten floor 52. They also allow the assembly 1 to utilize eitherreflected light, fluorescent emission or transmitted light in connectionwith the sample to achieve the desired results. The specific exampleshown in FIGS. 5 and 6 uses a single tube; however, multiple tubes canalso be analyzed by the assembly 1 by mounting a sample tube in adiametrically opposed position on the platen 3, and by altering thetiming from the index to the flash so as to provide readings for eachseparate tube. The drive shaft of the earlier described centrifuge motor13 is designated by the numeral 13′.

[0041]FIGS. 6 and 7 provide details of the manner in which the motordrive shaft 13′ is connected to the platen 3; and also details of themanner in which the sample tube 9 is connected to the platen hub 54. Themotor drive shaft 13′ is affixed to a drive disc 68 which is disposedinside of the hub 54. The disc 68 has a pair of drive pins 70 securedthereto, which drive pins 70 project through the hub windows 58. Thedrive pins 70 provide the sole driving contact between the motor driveshaft 13′ and the platen 3. Rotation of the disc 68 by the motor driveshaft 13′ causes the pins 70 to engage the sides of the hub windows 58thereby causing the hub 54 and the platen 3 to rotate with the disc 68.A rod 72 is rotatably mounted in the hub 54. The rod 72 includes acollar 74 at an end thereof which collar 74 receives one end of thesample tube 9. The collar 74 houses a resilient O-ring (not shown) whichgrips the end of the tube 9. A toothed ratchet 76 is mounted on the rod72 and is operable to cause stepwise selective rotation of the collar 74and sample tube 9 in the following manner. A spring-biasedratchet-engaging pawl 78 is mounted on the disc 68, and aratchet-engaging blade spring 80 is mounted on the hub cover 56. Whenthe centrifuge motor drive shaft 13′ is being powered by the motor 13,rotation of the disc 68 moves the pawl 78 into engagement with one ofthe teeth on the ratchet 76 and the blade spring 80 moves down intoengagement with a diametrically opposed tooth on the ratchet 76, asshown in FIG. 7. In order to selectively rotate the ratchet 76 andsample tube 9, power to the centrifuge motor 13 is periodicallyinterrupted so as to momentarily slow rotation of the drive shaft 13′.The momentum of the platen 3 causes it, and its hub 54, to momentarilyrotate at a faster rate than the disc 68 so as to disengage the pawl 78from the ratchet 76 and to disengage the drive pins 70 from the platenhub 54. During this momentary disengagement, the pawl 78 will disengagefrom the ratchet tooth, and move to a position wherein it engages thenext adjacent ratchet tooth. The motor 13 is then re-energized to fullspeed, causing the drive pins 70 to re-engage the hub 54 and causing thepawl 78 to impart a clockwise rotation step of the ratchet 76 and thesample tube 9. When the pawl 78 thus drives the next adjacent ratchettooth, rotation of the ratchet 76 will cause the spring 80 to engage adiametrically opposed next adjacent tooth on the ratchet 76 thusstabilizing the ratchet 76 and the sample tube 9 in the new rotationalposition. Stepwise rotation of the sample tube 9 thus allows the imagedissector 36 to “see” the entire periphery of the sample in the tube 9as the latter is being centrifuged. Circumferential variations in theposition of the descending sample component interfaces 8 will thus betaken into account by the system. The aforesaid pawl and ratchet tuberotating mechanism is the invention of Michael R. Walters of BectonDickinson and Company, and is described in this application for thepurpose of satisfying the “best mode” requirements of the patentstatute.

[0042]FIG. 8 is a plan view of the centrifuge platen 1 showing thesample tube 9 and a detectable target reference device 25 which isdisposed on the platen 1 and angularly offset from the tube 9 by aprecisely known angle Ø. The device 25 is preferably formed from amaterial such as a fluorescent plastic film which will fluoresce orreflect light in a manner similar to that of the tube 9. The device 25preferably has a plurality of lines or bands 27 which are perpendicularto the axis of the device 25. The device 25 is preferably formed from astable fluorescent or reflective fluorescent plastic film material sothat the energy emitted from the device 25 can be used to calibrate theoptical system of the instrument.

[0043] It is well known that quantitative measurements of fluorescentenergy are difficult, in that the energy measured depends on theintensity of the excitation source, the temperature of the sample, andthe responsiveness of the detection system, all of which may vary due toa number of factors. Although it is very difficult to provide any typeof long term calibration, it is practical to periodically compare theenergy level response from a target such as the reference device 25,which response is stable over time, to the energy level response fromthe sample 9, which response may not be stable over time. When theenergy level response from the sample 9 appears to the instrument to below, a comparison of successive sample 9 energy level responses andreference device 25 energy level responses can be made. The comparisonof energy level responses can be used to determine whether the sample 9energy level response is low because of a characteristic of the sample9, or because of a system error in the instrument's fluorescenceresponse measurement system. If the latter cause of faulty signal levelmeasurements is detected, then the instrument's signal level measurementsystem will be recalibrated based on the known signal level measurementsreceived from the reference device 25.

[0044] Furthermore, the length of the bands 27 in the reference device25 can be sensed by the imaging system in the instrument, and since thelength of the bands 27 can be precisely delineated, they can be used forspatial calibration of the entire imaging system. One optical errorwhich might occur, for example, is that the imaging lens and filtersystem may project an image onto the detector 36 which is non-linear.Thus, equidistant spatial points at the center of the image may notcover the same detector area as those points at the edge of the image.This phenomenon is encountered in photography wherein it is referred toas a pincushion or “barrel” effect, and it prevents accurate spatialmeasurements unless it is remedied. If the equidistant bands 27 aremapped by the detector 36 across the entire image length, then anydistortion can be detected and compensated for in the finalcalculations.

[0045] In use, the centrifuge platen 1 is rotated in the directionindicated by the arrow A until it reaches operating speed. The pulsingof the flash tube 26 is adjusted by the microprocessor 17 until a clearimage of the target device 25 is obtained by the image dissector 36. Inpractice, the speed of the centrifuge is first calculated by using thetiming pulses given by the sensor 23. From this data, an assumed delaytime from the sensor pulse to the flash trigger is calculated based uponeither previously obtained times or a time which is preprogrammed intothe instrument when the instrument is assembled. Then a series of imagesof the target device 25 is captured at that “starting time” and otherstarting times which are both shorter and longer. The flash time delaywhich gives the best response from the device 25 is then chosen andimplanted into the system's operating software.

[0046] At this point, inasmuch as both the platen speed and the anglebetween the target device 25 and the tube 9 are known, the time delaybetween the device 25 and the detector 36 can be calculated, and thelatter time delay value is added to the time delay from the timing pulseto the detector 25, so as to provide a total time delay to be programmedinto the delay 22 for pulsing the flash tube 26 so as to preciselyilluminate the sample tube 9.

[0047] It will be appreciated that the aforesaid system and method ofoperation will suffice to synchronize the flash tube illumination pulseswith the passage of the sample tube past the flash tube at anycentrifuge platen rotational speed. The aforesaid procedure will haveutility, irregardless of aging of centrifuge components, system orsample operating temperatures, or similar operating conditionvariations. The fluorescent standard device disposed on the platen alsoallows the imaging system in the assembly to be calibrated andre-calibrated automatically in the field.

[0048] Since many changes and variations of the disclosed embodiment ofthe invention may be made without departing from the inventive concept,it is not intended to limit the invention otherwise than as required bythe appended claims.

What is claimed is:
 1. A method for timing momentary highlight-intensity illumination of a sample tube which is positioned on acentrifuge platen during centrifugation of the sample tube on theplaten, said method comprising: a) the step of providing a firstlocation on the centrifuge platen for receiving and holding the sampletube; b) the step of providing a sample tube-simulating device disposedon the platen in a second location which is offset from said firstlocation by a pre-determined included angle; c) the step of obtaining aclear image of said tube-simulating device during centrifugation of thesample tube on the centrifuge platen so as to establish a referencepoint in time when said tubesimulating device passes by a high intensitysample-illuminating component, which component is operably associatedwith the centrifuge platen; d) the step of measuring the rotationalspeed of the centrifuge platen through the use of a sensible elementassociated with the centrifuge platen; and e) the step of establishingand storing a time delay between said reference point in time when saidtube-simulating device passes by the high intensity sample-illuminatingcomponent and a second point in time when said first location on thecentrifuge platen passes by the sample-illuminating component as afunction of the platen rotational speed and the known included anglebetween the tube-simulating device and the first location on thecentrifuge platen whereby a clear image of the sample tube can beobtained by momentarily energizing said sample-illuminating component atsaid second point in time.
 2. The method of claim 1 wherein said sampletube-simulating device also includes a plurality of differentiallyfluorescent bands of known length which bands simulate fluorescentlayers of components in a sample contained in the sample tube.
 3. Themethod of claim 2 wherein said sample-illuminating component illuminatesthe sample tube at a first wavelength in the range of about 420 to about480 nm, and wherein said fluorescent bands fluoresce light in the rangeof about 530 to about 580 nm, and about 620 to about 680 nm.
 4. A methodfor calibrating an imaging system so as to obtain accurate imaging of alayered sample that is contained in a sample tube which is positioned ona centrifuge platen in a first location during centrifugation of thesample and tube, said method comprising: a) the step of spinning theplaten so as to commence gravimetric compaction of the sample into atleast one discernable sample layer in the sample tube; b) the step ofproviding a sample tube-simulating device that is disposed on the platenin a location which is offset from said first location, said sampletube-simulating device including at least one fluorescent band of knownlength, which band simulates a fluorescent layer of a component in asample contained in the sample tube; and c) the step of obtaining aclear image of said sample tube-simulating device during centrifugationof the sample tube on the centrifuge platen so as to establish at leastone known reference sample tube-simulating device band length for theimaging system for use in establishing at least one actual sample layerlength imaged by the imaging system during centrifugation of the sample.5. The method of claim 4 wherein said fluorescent band emits afluorescent signal in the range of about 530 to about 580nm, or in therange of about 620 to about 680 nm, when subjected to light in the rangeof about 420 to about 480 nm.
 6. A system for timing intermittantillumination of a whole blood sample, which sample is contained in atransparent sample tube, during centrifugation of the transparent sampletube, said system comprising: a) a centrifuge assembly including aplaten and a motor for rotating the platen, said platen including asupport for the sample tube during centrifugation of the blood sample;b) a high intensity light source for illuminating the tube duringcentrifugation of the tube on said platen; c) a first filter interposedbetween the light source and the tube for converting light emitted fromsaid light source to a wavelength which is in the range of between about420 to about 480 nm; d) a linear image dissector operatively associatedwith said centrifuge platen so as to create analog signals resultingfrom light rays emitted from the sample in the tube, said analog signalsbeing representative of signal values from a plurality of points alongthe tube, which when digitized, allow a microprocessor to locate andmeasure the distance between adjacent interfaces of the target componentlayer; e) a second filter set interposed between the image dissector andthe tube for converting light rays emitted from said sample andtransmitted to said image dissector to a first wavelength which is inthe range of between about 530 to about 560 nm, and to a secondwavelength which is in the range of between about 620 and about 680 nm;d) a digitizer connected to said image dissector for converting saidanalog signals to digital signals; e) a microprocessor connected to saiddigitizer for receiving said digital signals from said digitizer, saidmicroprocessor being operable to convert said digital signals into aquantification of the degree of compaction of said target constituentcomponent layer, and thereby the volume of said target constituentcomponent layer; f) a mechanism for periodically pulsing said lightsource so that the latter is activated only when the sample tube passesby said image dissector.
 7. A system for calibrating an imaging systemso as to obtain accurate sample layer imaging of constituent layers in awhole blood sample, which sample is contained in a transparent sampletube, during centrifugation of the transparent sample tube, said systemcomprising: a) a centrifuge assembly comprising a platen and a motor forrotating the platen, said platen including a support for the tube duringcentrifugation of the blood sample; b) a high intensity light source forilluminating the tube during centrifugation of the tube on said platen;c) a first filter interposed between the light source and the tube forconverting light emitted from said light source to a wavelength which isin the range of between about 420 to about 480 nm; d) a linear imagedissector operatively associated with said centrifuge platen so as tocreate analog signals resulting from light rays emitted from the samplein the tube, said analog signals being representative of signal valuesfrom a plurality of points along the tube, which when digitized, allow amicroprocessor to locate and measure the distance between adjacentinterfaces of the target component layer; e) a second filter setinterposed between the image dissector and the tube for converting lightrays emitted from said sample and transmitted to said image dissector toa first wavelength which is in the range of between about 530 to about560 nm, and to a second wavelength which is in the range of betweenabout 620 and about 680 nm; d) a digitizer connected to said imagedissector for converting said analog signals to digital signals; e) amicroprocessor connected to said digitizer for receiving said digitalsignals from said digitizer, said microprocessor being operable toconvert said digital signals into a quantification of the degree ofcompaction of said target constituent component layer, and thereby thevolume of said target constituent component layer; f) a mechanism forperiodically pulsing said light source so that the latter is activatedonly when the sample tube passes by said image dissector.